Immune system of an organism has been developed with surveillance and defense mechanism by recognition and elimination of pathogenic foreign microorganisms such as bacteria and viruses. Therefore, the organism distinguishes own cells or tissues (self-antigens) from foreign microorganism (nonself-antigens), and does not respond to the self-antigens, or respond to them having the failure to mount immune response. Accordingly, the organism has developed an acquired immunity to eliminate nonself-antigens immediately and efficiently.
T lymphocytes (T cells) and B lymphocytes (B cells) are the primary cells of the adaptive arm of the immune system. Both are involved in acquired immunity and the complex interaction of these cells is required for the expression of the full range of effector and memory cells of the immune responses. T cells are specific for foreign antigens and their number must increase enormously in response for specific host defense.
Optimum activation of T cell depends on two discrete receptor-ligand recognition events. The major event is the interaction of T cell receptors (TCRs) with peptide-major histocompatibility complexes (PMHC) that are displayed on the surface of the antigen-presenting cell (APC) such as B cell, macrophage and dendritic cell. However, in the absence of a co-stimulatory signal, the TCR-pMHC interaction alone is insufficient for complete T cell activation and may result in either apoptotic death or prolonged unresponsiveness of the responding T cell (Agrewala et al. 1994, 1998).
It is the interaction of a family of related co-stimulatory receptors with their respective ligands that furnishes the second co-stimulatory signals (CD28, CD40L), which are required for efficient T cell activation. Moreover, a second, complementary set of co-stimulatory signals (CTLA-4, PD-1, BTLA) also provide negative signals that reduce the immune response and as such function to maintain the peripheral T cell tolerance to protect against autoimmunity (Nishimura et al. 2001, Greenwald et al. 2001).
The main co-stimulatory molecules expressed on the surface of T cells are CD28 and CTLA-4/CD152. CD28 is constitutively expressed on T cells. CD28 ligation enhances the magnitude and duration of T-cell responses; induces the anti-apoptotic gene BCL-XL; increases cytokine secretion, particularly interleukin 2 (IL-2); enhances cell adhesion; facilitates reorganization of the T-cell plasma membrane upon binding to an APC; prevents anergy induction; and supports germinal center formation (Lanzavecchia et al. 1999). CD28 co-stimulation is necessary for the initiation of most T cell responses, and blockade of CD28 signaling results in a greatly reduced ability to respond to protein antigens, parasites and some viruses, and to generate germinal centers and mediate B-cell help. This has therapeutic implications; in that blockade of CD28 co-stimulation can be profoundly immunosuppressive, preventing induction of pathogenic T cell responses in autoimmune disease models and allowing for prolonged acceptance of allograft in models of organ transplantation (Salomon et al. 2001).
CTLA-4 (CD152) mediates such an inhibitory signal. CTLA-4 cross-linking by immobilized mAb or by soluble antibody cross-linked with a secondary antibody inhibited T cell responses induced by anti-CD3 and anti-CD28 antibodies (Krummel et al. 1996). Although CTLA-4 displays the common features of the CD28 family members, it is unique in several important ways. First, CTLA-4 has a markedly higher affinity for shared ligands B7-1 and B7-2 compared with CD28 (Kd 0.2-0.4 ηm versus 4.0 μm), and a 40-100 fold higher avidity (van der Merwe et al. 1997).
Secondly, CTLA-4 has a unique expression pattern. Unlike CD28, CTLA-4 is not expressed constitutively on the cell surface of naïve T cells. CTLA4 is only expressed after the CD4+ T cell becomes activated (2-3 days post APC-TCR engagement) and upon engagement with B7 molecules, transduces a negative signal to T cells. As the binding affinity of B7-1 and B7-2 for CTLA4 is 40-50× greater than for CD28, negative signaling would dominate on activated T cells, thereby terminating the immune response. CTLA-4 blockade in vivo enhances antigen-specific and anti-parasite responses, tumor rejection, autoimmune disease, and exacerbates graft rejection (Tivol et al. 1996, Chambers et al. 2001). In vitro, engagement of CTLA-4 results in inhibition of T cell proliferation, cytokine production and cell cycle progression (Chambers et al. 2001, Freeman et al. 2000).
CTLA-4 regulates peripheral tolerance by a number of different mechanisms. First, CTLA-4 regulates the activation of T cells by directly modulating T cell receptor signaling (i.e. TCR chain phosphorylation) (Lee et al. 1998) as well as biochemical signals (i.e. ERK activation). Second, recent studies have shown that the CD4+ CD25+ immunoregulatory T cells constitutively express CTLA-4 (Salomon et al. 2000). In fact, signaling via CTLA-4 is essential for the function of these cells (Takahashi et al. 2000). Thus, CTLA-4 may regulate signal transduction in the cells, which leads to differentiation into regulatory T cells; or alternatively, CTLA-4 engagement on the effector cells may alter signal transduction and subsequent cytokine production. Cross-linking of CTLA-4 induces secretion of the immunoregulatory TGF-β cytokine (Chen et al. 1998), which provides one possible mechanism of action for the CD4+CTLA-4+CD25+ regulatory T cells.
Although the findings appear to suggest multiple functional effects of CTLA-4 in altering immune function, one of the models says that these apparently different activities are all related and the major effect of CTLA-4 is to alter the threshold of T cell activation by altering early events in TCR signaling. In fact, it has been demonstrated that treatment of T cells with cyclosporin A, a calcineurin inhibitor that modulates calcium mobilization, leads to the generation of a TGFβ-producing T cells that are similar to the CD4+CTLA-4+CD25+ regulatory T cells (Prashar et al. 1995). Thus, the effects of CTLA-4 engagement whether directed at the inhibition of CD28 signaling, modulation of proximal TCR signals or down-stream effector pathways of T cell activation result in altered T cell differentiation and down regulation of immune responses. Hence, there exists a possibility of therapeutic potential of suppressing the exacerbation of diseases by regulating the expression of CTLA-4/CD28 on the surface of T cells by Caerulomycin A.
Many co-stimulatory molecules expressed on the surface of antigen presenting cells are known to date but B7-1 and B7-2 are the most potent and are responsible for the activation of T cells. Their interaction with CD28/CTLA-4 receptors expressed on T cell surfaces is quite crucial. Binding of CD28 to its ligands B7-1 and B7-2, delivers a co-stimulatory signal to T cell, enhancing their proliferation and cytokine secretion and preventing the induction of T cell anergy (Linsley et al. 1991). In contrast, the engagement of CTLA-4 by these same ligands results in down-regulation of the response that is essential for maintaining T cell homeostasis and self-tolerance (Tivol et al. 1995). B7-1 and B7-2, which share ˜25% sequence identity, are type I transmembrane glycoproteins (Stamper et al. 2001). It is established phenomenon that interaction of CD28 and CTLA-4 with B7-ligands is critical for activation and inhibition of immune responses and tolerance respectively (Greenwald et al. 2005).
In summary, B and T cell responses depend on multiple and complex interdependent events. Because of the key role of B and T cell in immunity, their regulation is a major target for treating and/or preventing a large variety of diseases that require or benefit from an enhanced or reduced immunity, e.g. autoimmune diseases including type I diabetes, multiple sclerosis, asthma, arthritis, myasthenia gravis, lupus erythematosus, psoriasis, colitis, or rejection of transplanted organs, or immuno-deficiency diseases, and cancer. Therefore, there is a strong need for drugs capable of modulating the complex B and T cell responses for the purpose of treating and preventing numerous immunological disorders and diseases.
Successful organ transplantation requires effective physiological and pharmacological intervention of the immune system of an organ recipient. One approach to intervention of immune response in an organ transplant recipient, especially a recipient targeted for an allogenic graft, is by the use of immunosuppressive drugs. These drugs are used to prolong survival of transplanted organs in recipients in cases involving, for example, transplants of kidney, liver, heart, lung, bone marrow and pancreas.
There are several types of immunosuppressive drugs available for use in reducing organ rejection in transplantation. Such drugs fall within three major classes, namely: antiproliferative agents, anti inflammatory compounds and inhibitors of lymphocyte activation.
Examples of the class of cytotoxic or antiproliferative agents are azathioprine, cyclophosphamide and methotrexate. Drugs of the antiproliferative class may be effective immunosuppressives in patients with chronic inflammatory disorders and in organ transplant recipients by limiting cell activation and proliferation. These drugs which abrogate mitosis and cell division have severe cytotoxic side effects on normal cell populations which have a high turn-over rate, such as bone marrow cells and cells of the gastrointestinal (GI) tract lining. Accordingly, such drugs often have severe side effects, particularly, lymphopenia, neutropenia, bone marrow depression, hemorrhagic cystitis, liver damage, increased incidence of malignancy, hair loss, GI tract disturbances, and infertility.
A second class of immunosuppressive drugs for use in transplantation is provided by compounds having anti-inflammatory action. Representatives of this drug class are generally known as adrenal corticosteroids and have the advantage of not exerting globally systemic cytotoxic effects. These compounds usually act by preventing or inhibiting inflammatory responses, cytokine production, chemotaxis, neutrophil, macrophage or lymphocyte activation, or their effector function. Typical examples of adrenal corticosteroids are prednisone and prednisolone, which affect carbohydrate and protein metabolism as well as immune functions. Compounds of this class are sometimes used in combination with cytotoxic agents, such as compounds of the antiproliferative class because the corticosteroids are significantly less toxic. But the adrenal corticosteroids lack specificity of effect and can exert a broad range of metabolic, anti-inflammatory and immune effects. Typical side effects of this class include increased organ-recipient infections and interference with wound healing, as well as disturbing hemodynamic balance, carbohydrate and bone metabolism and mineral regulation.
A third class of immunosuppressive drugs for use in organ transplantation is provided by compounds, which are immunomodulatory and generally prevent or inhibit leukocyte activation. Such compounds usually act by blocking activated T-cell effector functions or proliferation, or by inhibiting cytokine production, or by preventing or inhibiting activation, differentiation or effector functions of platelet, granulocyte, B-cell, or macrophage actions. The cyclosporin family of compounds is the leading example of drugs in this class. Such compounds are polypeptide fungal metabolites, which have been found to be very effective in suppressing helper T-cells so as to reduce both cellular and humoral responses to newly encountered antigens. Cyclosporins alter macrophage and lymphocyte activity by reducing cytokine production or secretion and, in particular, by interfering with activation of antigen-specific CD4 cells, by preventing IL-2 secretion and secretion of many T-cell products, as well as by interfering with expression of receptors for these lymphokines on various cell types. Cyclosporin A, in particular, has been used extensively as an immunosuppressive agent in organ transplantation. Other microbial metabolites include cyclosporins such as cyclosporin B and cyclosporin G, and another microbial product known as FK-506. Cyclosporin A suppresses humoral immunity as well as cell-mediated reactions. Cyclosporin A is for organ rejection in kidney, liver, heart, pancreas, bone-marrow and heart-lung transplants. Cyclosporin A is also useful in the treatment of autoimmune and inflammatory diseases, including rheumatoid arthritis, Crohn's disease, Graves' disease, severe psoriasis, aplastic anemia, multiple-sclerosis, alopecia areata, penphigus and penphigoid, dermatomyositis, polymyositis, Behcet's disease, uveitis, pulmonary sarcocidiosis, biliary cirrhosis, myasthenia gravis and atopic dermatitis.
Cyclosporins possess several significant disadvantages. While cyclosporins have provided significant benefits in organ transplantation, cyclosporins are non-specific immunosuppressives. Desirable immune reactions may be reduced against foreign antigens. Tolerated dosages do not provide complete suppression of rejection response. Thus, immunologic reactions to transplanted tissue are not totally impeded, requiring concomitant treatment with prednisone, methylprednisolone, and/or other immunosuppression agents, including monoclonal antibodies such as anti-CD3 or anti-CD5/CD7. Cyclosporins can produce severe side effects in many organ recipients, and show host-variable effects on the liver, kidney, the central nervous system and gastro-intestinal tract. Significant among the adverse side effects are damage to the kidney and liver, hyperplasia of gum tissue, refractory hypertension and increased incidence of infections and malignancy.
Thus, the need remains for efficacious and selective immunosuppressive drugs for the treatment of autoimmune diseases and also in organ transplantation, especially for grafts between less-than-perfectly matched donor-recipient pairs. We therefore, present a proposal that takes a rationale approach to utilize Carulomycin isolated from the novel species of actinomycetes as an immunosuppressant for suppressing immune response. This and other objectives of the present invention, as well as additional inventive features, will be apparent from the detailed description provided herein. The inventors of the present invention have established that the optimum dosage of Caerulomycin A required in vivo was 5.0 mg/kg/body wt. The dosage of Caerulomycin A used in vitro experiments for inducing inhibition in the proliferation was 10 times lesser than the Cyclosporin A, which is a known immunosuppressant.